New Monocyclic, Bicyclic, and Tricyclic Ethynylcyanodienones as Activators of the Keap1/Nrf2/ARE Pathway and Inhibitors of Inducible Nitric Oxide Synthase

Article abstract of DOI:10.1021/acs.jmedchem.5b00393

A monocyclic compound 3 (3-ethynyl-3-methyl-6-oxocyclohexa-1,4-dienecarbonitrile) is a highly reactive Michael acceptor leading to reversible adducts with nucleophiles, which displays equal or greater potency than the pentacyclic triterpenoid CDDO in inflammation and carcinogenesis related assays. Recently, reversible covalent drugs, which bind with protein targets but not permanently, have been gaining attention because of their unique features. To explore such reversible covalent drugs, we have synthesized monocyclic, bicyclic, and tricyclic compounds containing 3 as an electrophilic fragment and evaluated them as activators of the Keap1/Nrf2/ARE pathway and inhibitors of iNOS. Notably, these compounds maintain the unique features of the chemical reactivity and biological potency of 3. Among them, a monocyclic compound 5 is the most potent in these assays while a tricyclic compound 14 displays a more robust and specific activation profile compared to 5. In conclusion, we demonstrate that 3 is a useful electrophilic fragment for exploring reversible covalent drugs. (Chemical Equation Presented).

Full text of DOI:10.1021/acs.jmedchem.5b00393

Article
pubs.acs.org/jmc
New Monocyclic, Bicyclic, and Tricyclic Ethynylcyanodienones as
Activators of the Keap1/Nrf2/ARE Pathway and Inhibitors of
Inducible Nitric Oxide Synthase
Wei Li,† Suqing Zheng,†,# Maureen Higgins,‡ Rocco P. Morra, Jr.,† Anne T. Mendis,† Chih-Wei Chien,†
Iwao Ojima,†,§ Dale F. Mierke,∥ Albena T. Dinkova-Kostova,‡,⊥ and Tadashi Honda*,†,§
†Institute of Chemical Biology and Drug Discovery, Stony Brook University, Stony Brook, New York 11794, United States
‡Division of Cancer Research, Medical Research Institute, University of Dundee, Dundee DD1 9SY, Scotland United Kingdom
§Department of Chemistry, Stony Brook University, Stony Brook, New York 11794, United States
∥Department of Chemistry, Dartmouth College, Hanover, New Hampshire 03755, United States
⊥Department of Pharmacology and Molecular Sciences, Johns Hopkins University School of Medicine, Baltimore, Maryland 21205,
United States
S
* Supporting Information
ABSTRACT: A monocyclic compound 3 (3-ethynyl-3-
methyl-6-oxocyclohexa-1,4-dienecarbonitrile) is a highly re-
active Michael acceptor leading to reversible adducts with
nucleophiles, which displays equal or greater potency than the
pentacyclic triterpenoid CDDO in inflammation and carcino-
genesis related assays. Recently, reversible covalent drugs,
which bind with protein targets but not permanently, have
been gaining attention because of their unique features. To explore such reversible covalent drugs, we have synthesized
monocyclic, bicyclic, and tricyclic compounds containing 3 as an electrophilic fragment and evaluated them as activators of the
Keap1/Nrf2/ARE pathway and inhibitors of iNOS. Notably, these compounds maintain the unique features of the chemical
reactivity and biological potency of 3. Among them, a monocyclic compound 5 is the most potent in these assays while a tricyclic
compound 14 displays a more robust and specific activation profile compared to 5. In conclusion, we demonstrate that 3 is a
useful electrophilic fragment for exploring reversible covalent drugs.
known to date.1−4 Compound 1 suppresses pro-inflammatory
responses and induces heme oxygenase-1 (HO-1) in
RAW264.7 cells, upregulates NAD(P)H:quinone oxidoreduc-
1. INTRODUCTION
Tricyclic compound 1 [TBE-31, ( )-(4bS,8aR,10aS)-10a-
ethynyl-4b,8,8-trimethyl-3,7-dioxo-3,4b,7,8,8a,9,10,10a-octahy-
tase 1 (NQO1) in Hepa1c1c murine hepatoma cells through
drophenanthrene-2,6-dicarbonitrile, Figure 1] is one of the
the Keap1/Nrf2/ARE pathway, and protects animals against
liver carcinogenesis induced by aflatoxin.3 Incorporation of
most potent activators of the Keap1/Nrf2/ARE pathway
small quantities (9.2 mg per kg of food) of 1 in the diet of mice
profoundly and dose dependently induces NQO1 and
glutathione S-transferases in the stomach, skin, and liver.4
Long-term (5 days per week for 4 weeks) topical daily
applications of 200 nmol of 1 causes a robust systemic
induction of the Keap1/Nrf2/ARE pathway and decreases the
6-thioguanine incorporation into DNA of skin, blood, and liver
of azathioprine-treated mice, indicating extraordinary bioavail-
ability and efficacy.5
Tricyclic compound 1 has two different monocyclic non-
enolizable cyanoenones 2 and 3 (Figure 1) in rings A and C.
We chemically demonstrated by UV spectroscopy that the A
and C rings of TBE-31 produce reversible Michael adducts with
the sulfhydryl groups of Keap1 and dithiothreitol (DTT).3,4 We
Figure 1. Structures of 1 (TBE-31), monocyclic cyanoenones 2 and 3,
CDDO, and bardoxolone methyl.
Received: March 10, 2015
Published: May 12, 2015
© 2015 American Chemical Society
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speculated that 1 regulates proteins affecting inflammation,
oxidative stress, differentiation, apoptosis, and proliferation,
including Keap1, IKKβ, and JAK1, to name a few, by reversible
Michael addition between the cyanoenone functionalities and
the sulfhydryl groups of cysteine moieties on these proteins.
More recently, we have focused on monocyclic cyanoenones,
which are considered to be the phamacophores of 1. We
designed and synthesized a preliminary set of eight monocylic
cyanoenones, including 2 and 3, and then evaluated the
chemical reactivity as Michael acceptors and biological
potency.6 Among monocyclic cyanoenones, ethynylcyanodie-
none 3 is a highly reactive Michael acceptor with thiol
nucleophiles. Furthermore, an important feature of 3 is that its
Michael addition is reversible. For the induction of NQO1, 3
demonstrates the highest potency of this series.6 For the
inhibition of nitric oxide (NO) production in RAW264.7 cells
(murine macrophage-like cell line) stimulated with IFN-γ, 3
also shows the highest potency.6 Remarkably, in this assay, the
ethynylcyanodienone 3, which has such a simple structure, is
about three times more potent than a pentacyclic triterpenoid,
CDDO, whose methyl ester (bardoxolone methyl) is presently
being evaluated in phase 2 clinical trials for the treatment of
pulmonary arterial hypertension (PAH) in the United States
and diabetic nephropathy in Japan. Overall, we found that 3
displays unique features regarding chemical reactivity and
biological potency.
the reversible covalent adducts with protein targets. On the
basis of the evidence described above, ethynylcyanodienone 3 is
considered to be one of the best fragments that we can use for
exploring reversible covalent drugs which are targeting on the
Keap1/Nrf2/ARE pathway. Thus, we have designed and
synthesized new monocyclic, bicyclic, and tricyclic compounds
I−III containing 3 as the electrophilic fragment (Figure 2,
Figure 2. Monocyclic, bicyclic, and tricyclic compounds containing
ethynylcyanodienone 3.
Generally, there are three categories of drugs: (1) irreversible
covalent drugs, which permanently bind to their protein targets
through covalent bonds. This category includes alkylating
agents (e.g., cyclophosphamide, mitomycin C), β-lactam
antibiotics (e.g., penicillin, cephalosporin) and irreversible
enzyme inhibitors (e.g., acetylsalicylic acid, omeprazole); (2)
reversible noncovalent drugs, which noncovalently (e.g.,
hydrogen bond, hydrophobic effects, and van der Waals forces)
bind with protein targets; this category contains receptor
antagonists (for example, angiotensin II receptor blocker, β-
blockers, and histamine H2-receptor antagonists and many
others); (3) reversible covalent drugs, which covalently bind
but not permanently with protein targets. This final category is
the newest and gaining the most interest because of the
resulting enhanced properties. Currently, dimethyl fumarate
(DMF),7 a Michael acceptor and an activator of the Keap1/
Nrf2/ARE pathway, is the only drug that is clinically used for
the treatment of multiple sclerosis. CDDO and bardoxolone
methyl also belong to this category. Recently, the development
of a series of reversible covalent inhibitors of MSK/RSK-family
kinases has been reported.8
Although irreversible covalent drugs have long duration of
action, high potency, and high ligand efficiency because they
irreversibly bind to both on- and off-protein targets, there is a
potential for immune-mediated hypersensitivity and therefore
such drugs are not suitable for chronic dosing. Reversible
noncovalent drugs do not form permanent adducts and are
therefore suitable for chronic dosing. However, their selectivity
and potency are moderate because their ligand efficiency is
usually poor. In sharp contrast, reversible covalent drugs have
high potency, high ligand efficiency, and long duration of action
and because they do not form permanent adducts, they are
suitable for chronic dosing. Overall, reversible covalent drugs
combine the advantages and circumvent the disadvantages of
irreversible covalent and reversible noncovalent drugs.
specifically Table 1). Because several monocyclic cyanoenones6
have been synthesized and biologically evaluated and
consequently they gave interesting SARs, we have designed
I−II. Also, we have designed III because tricyclic compounds
which do not contain aromatic rings in the mother nuclei have
never been synthesized although more than 40 tricyclic
compounds have been synthesized so far.2 Then we measured
the reactivity of ethynylcyanodienone moiety as a Michael
acceptor in these compounds I−III by UV spectroscopy and
evaluated their potency for the induction of NQO1 in
Hepa1c1c murine hepatoma cells and the inhibition of iNOS
in RAW264.7 cells stimulated with LPS. We herein describe the
full account of our synthetic work with these new compounds,
their unique and interesting features with respect to the
chemical reactivity as Michael acceptors, and biological
potency.
2. CHEMISTRY
2.1. Monocyclic Ethynylcyanodienones. Monocyclic
diethynylcyanodienone 4 was synthesized in nine steps from
2,2-dimethyl-1,3-dioxan-5-one by the sequence as shown in
Scheme 1. Known dienophile precursor 169 was synthesized in
42% yield through Sonogashira coupling between triisopro-
pylsilylacetylene (TIPS acetylene) and the triflate derived from
2,2-dimethyl-1,3-dioxan-5-one. Diels−Alder reaction between
the Danishefsky’s diene and 16 gave previously reported adduct
17.9 Without purification of the crude adduct, 17 was treated
with (chloromethyl)triphenylphosphonium chloride10 in the
presence of n-BuLi in THF to afford 18. Dehydrochlorination
of 18 with LDA in THF, followed by quenching the acetylide
with TMSCl, produced 19.11 Monocyclic enone 20 was
obtained in pure form by the treatment of 19 with TFA in
1,2-dichloroethane, followed by flash column chromatography
purification (33% yield from 16). Cyanation of the enolate of
20 generated using LDA in THF, with p-TsCN, gave 21 in 77%
Nevertheless, reversible covalent drugs have been largely
ignored because of the lack of reactive compounds to produce
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a
Scheme 1
a
Reagents: (a) triflic anhydride, DMAP, pyridine; (b) TIPS acetylene, Pd(PPh3)4, CuI, Et2NH; (c) toluene; (d) Ph3PCH2Cl2, n-BuLi, THF; (e)
TMSCl, LDA, THF; (f) TFA, ClCH2CH2Cl; (g) p-TsCN, LDA, THF; (h) TBAF, THF; (i) PhSeCl, pyridine, CH2Cl2, 30% aqueous H2O2, CH2Cl2.
yield.12 Deprotection of 21 with TBAF in THF, followed by
addition of PhSeCl in the presence of pyridine and subsequent
oxidation/elimination of the selenated intermediate with 30%
aqueous H2O2 solution afforded 4 in 57% yield.13
Monocyclic ethynylvinylcyanodienone 5 was synthesized in
five steps starting from adduct 17 (Scheme 2). A Wittig
produced in three steps from 27 by the same sequence (Wittig
reaction, conversion of a chlorovinyl group to a TMS protected
ethynyl group, and deprotection) as for 20 (21% yield from
26). Cyanation of the enolate of 30 with p-TsCN, followed by
deprotection with TBAF, gave 32 in 29% yield. Cyanodienone
6 was prepared in 79% yield from 32 by addition of PhSeCl in
the presence of pyridine and subsequent oxidation/elimination
of the selenated intermediate with 30% aqueous H2O2 solution.
2.2. Bicyclic Ethynylcyanodienones. Bicyclic ethynylcya-
nodienone 7 was synthesized in 10 steps from the previously
reported compound 33,15 which was prepared by Michael
addition between methyl 2-oxocyclopentanecarboxylate and
but-3-en-2-one (Scheme 4). Intramolecular aldol condensation
of 33 in the presence of pyrrolidine (1 equiv) and acetic acid (1
equiv) in EtOAc gave enone 34 in 58% yield. Enone 34 was
protected with ethylene glycol in the presence of PPTS in
toluene to afford 35 in 95% yield. Reduction of 35 with LAH in
Et2O, followed by Swern oxidation, produced 36. Without
purification, 36 was treated with Ohira reagent (dimethyl(1-
diazo-2-oxopropyl)phosphonate)16 to give 37 (48% yield from
35). The lithium acetylide, which was derived from 37 with
MeLi, was protected with TBSCl to afford 38 in 92% yield.
Removal of a ketal of 38 under acidic conditions, followed by
cyanation of the lithium enolate of 39 with p-TsCN, provided
40 (47% yield from 38). The desired compound 7 was
obtained by deprotection of 40 with TBAF in THF, followed
by DDQ oxidation in PhH (22% yield from 40).
Bicyclic ethynylcyanodienone 8, which has a nonenolizable
cyanodienone while 7 has an enolizable cyanodienone, was
synthesized in 11 steps from the known compound 42,17 which
was obtained by methoxycarbonylation of 2,2-dimethylcyclo-
pentanone (Scheme 5). Enone 43 was prepared by intra-
molecular aldol condensation of the adduct obtained by
Michael addition between 42 and but-3-en-2-one (53%
yield). Conversion from 43 to 46 was achieved by the same
sequence (ketalization, reduction, oxidation, and introduction
of an ethynyl group) as for 37 (23% yield from 43). A cyano
group was introduced using Johnson isoxazole method18
instead of p-TsCN. Removal of a ketal of 46, followed by
formylation with ethyl formate in the presence of NaH in THF,
gave 47. Without purification, 47 was treated with hydroxyl-
amine hydrochloride in aqueous EtOH to afford isoxazole 48
(46% yield from 46).18 The desired compound 8 was obtained
a
Scheme 2
a
Reagents: (a) Ph3PCH3I, n-BuLi, THF; (b) D-(+)-CSA, dioxane; (c)
p-TsCN, LDA, THF; (d) TBAF, THF; (e) PhSeCl, pyridine, CH2Cl2,
30% aqueous H2O2, CH2Cl2.
reaction on 17 with methyltriphenylphosphonium iodide in the
presence of n-BuLi in THF provided 22. Enone 23 was
obtained by the treatment of 22 with D-(+)-camphor sulfonic
acid [D-(+)-CSA] in 1,4-dioxane under reflux conditions (35%
yield from 17). The desired compound 5 was prepared in three
steps in 40% yield via 24 and 25 from 23 by the same sequence
(cyanation, deprotection, and insertion of a double bond) as for
4.
Monocyclic ethylethynylcyanodienone 6 was synthesized
with six steps from the Diels−Alder adduct 27, which was
obtained from the Danishefsky’s diene and 2-ethylacrolein (26)
under microwave conditions (Scheme 3).14 Enone 30 was
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a
Scheme 3
a
Reagents: (a) microwave at 120 °C; (b) Ph3PCH2Cl2, n-BuLi, THF; (c) TMSCl, LDA, THF; (d) TFA, CHCl3; (e) p-TsCN, LDA, THF; (f)
TBAF, THF; (g) PhSeCl, pyridine, CH2Cl2, 30% aqueous H2O2, CH2Cl2.
a
Scheme 4
a
Reagents: (a) pyrrolidine, AcOH, EtOAc; (b) ethylene glycol, PPTS, toluene; (c) LAH, Et2O; (d) (COCl)2, DMSO, CH2Cl2; (e) Ohira reagent,
K2CO3, MeOH; (f) MeLi, TBSCl, THF; (g) 10% aqueous HCl, MeOH; (h) p-TsCN, LDA, THF; (i) TBAF, THF; (j) DDQ, PhH.
a
Scheme 5
a
Reagents: (a) methyl vinyl ketone, Et3N; (b) pyrrolidine, AcOH, toluene; (c) ethylene glycol, PPTS, toluene; (d) LAH, Et2O; (e) (COCl)2,
DMSO, CH2Cl2; (f) Ohira reagent, K2CO3, MeOH; (g) 10% aqueous HCl, MeOH; (h) HCO2Et, NaH, THF; (i) NH2OH·HCl, aqueous EtOH; (j)
NaOMe, MeOH; (k) DDQ, PhH.
by cleavage of the isoxazole moiety of 48 with NaOMe in
MeOH,18 followed by DDQ oxidation in PhH (46% yield).
Bicyclic ethynylcyanodienone 9, which has a naphthalene
skeleton, was synthesized via the known compound 51 from
1,2,3,4-tetrahydronaphthalene-1-carboxylic acid (49) as shown
in Scheme 6. We have obtained racemic 51 by a new sequence
that is different from the previously reported synthesis19,20 of
optically active 51. Methyl ester 5021 was prepared by
methylation of 49, followed by insertion of a methyl group
(79% yield). Reduction of 50 with LAH and subsequent Swern
oxidation gave 51 (100% yield). A formyl group of 51 was
converted to an ethynyl group of 52 with Ohira reagent (87%
yield). Chromium-mediated allylic oxidation22 of 52 provided
ketone 53 in 52% yield. Formylation of 53 with ethyl formate
in the presence of NaOMe in PhH,23 followed by isoxazole
formation with hydroxylamine hydrochloride in aqueous EtOH,
afforded 55 in 84% yield. The desired compound 9 was
obtained by cleavage of the isoxazole moiety of 55 with
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a
Scheme 6
a
Reagents: (a) p-TsOH, MeOH; (b) MeLi, LDA, THF; (c) LAH, THF; (d) (COCl)2, DMSO, CH2Cl2; (e) Ohira reagent, K2CO3, MeOH; (f)
CrO3, t-BuO2H, CH2Cl2; (g) HCO2Et, NaOMe, PhH; (h) NH2OH·HCl, aqueous EtOH; (i) NaOMe, MeOH, Et2O; (j) PhSeCl, pyridine, CH2Cl2,
30% aqueous H2O2, CH2Cl2.
a
Scheme 7
a
Reagents: (a) NaH, Me2CO3; (b) methyl vinyl ketone, NaOMe, MeOH; (c) ethylene glycol, p-TsOH, toluene; (d) LAH, Et2O; (e) (COCl)2,
DMSO, CH2Cl2; (f) Ohira reagent, K2CO3, MeOH; (g) 10% aqueous HCl, MeOH; (h) HCO2Et, NaOMe, PhH; (i) NH2OH·HCl, aqueous EtOH;
(j) NaOMe, MeOH, Et2O; (k) PhSeCl, pyridine, CH2Cl2, 30% aqueous H2O2, CH2Cl2.
a
NaOMe in a mixture of MeOH and Et2O, followed by addition
of PhSeCl in the presence of pyridine and subsequent
oxidation/elimination of the selenated intermediate with 30%
aqueous H2O2 solution (92% yield).
Scheme 8
2.3. Tricyclic Ethynylcyanodienones. Tricyclic ethynyl-
cyanodienone 10, which has a phenanthrene skeleton, was
synthesized in 11 steps from 1-tetralone (Scheme 7). The
known compound 5624 was prepared by an improved
methodology than that previously reported. Methoxycarbony-
lation of 1-tetralone, followed by Robinson annulation with
ethyl vinyl ketone, gave 56 in 75% yield. After protection of an
enone of 56 with ethylene glycol (93% yield), a methox-
ycarbonyl group of 57 was converted to a formyl group of 58
by reduction with LAH and subsequent Swern oxidation (67%
yield). Transformation of a formyl group of 58 to an ethynyl
group with Ohira reagent afforded 59 in 74% yield. Isoxazole
60 was prepared in 73% yield from 59 by deketalization under
acidic conditions, followed by formylation with ethyl formate
and subsequent isoxazole formation with hydroxylamine
hydrochloride. The desired compound 10 was obtained by
isoxazole cleavage of 60 with NaOMe, followed by addition of
PhSeCl in the presence of pyridine and subsequent oxidation/
elimination of the selenated intermediate with 30% aqueous
H2O2 solution (82% yield).
a
Reagents: (a) NaH, Me2CO3; (b) methyl vinyl ketone, NaOMe,
MeOH; (c) 2,3-butanediol, p-TsOH, toluene; (c′) 2,3-butanediol,
PPTS, toluene; (d) LAH, Et2O; (e) (COCl)2, DMSO, CH2Cl2; (f)
Ohira reagent, K2CO3, MeOH; (g) 10% aqueous HCl, MeOH; (h)
HCO2Et, NaOMe, PhH; (i) NH2OH·HCl, aqueous EtOH; (j)
NaOMe, MeOH, Et2O; (k) PhSeCl, pyridine, CH2Cl2, 30% aqueous
H2O2, CH2Cl2.
Chloro- and bromotricyclic ethynylcyanodienones 11 and 12
were synthesized in 11 steps from 8-chloro- and 8-bromo-1-
tetralone (61 and 64)25 as shown in Scheme 8. Tricyclic
precursors 62 and 65 were obtained by methoxycarbonylation
of 61 and 64 with Me2CO, followed by Robinson annulation
with methyl vinyl ketone, respectively. Initially, although an
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a
enone of 62 was protected with ethylene glycol, because the
ketal was easily converted to the enone, the desired ketal was
not isolated. Consequently, we attempted to protect the enone
of 62 with a bulky diol, 2,3-butandiol in the presence of p-
TsOH in toluene, because it is known that such a bulky ketal
resists hydrolysis under acidic conditions.1 As we expected, the
more stable ketal 63 was obtained. Similarly, ketalization of 65
with 2,3-butandiol in the presence of PPTS in toluene gave 66
in 89% yield. The desired chloro- and bromotricyclic
compounds 11 and 12 were prepared in eight steps from 63
and 66 by the same sequence as for 10, respectively (11, 26%
yield from 62; 12, 33% yield from 66).
Scheme 10
Methyltricyclic ethynylcyanodienone 13 was synthesized in
10 steps from 62 (Scheme 9). We have employed a Buchwald
a
Scheme 9
a
Reagents: (a) NaNO2, 15% aqueous HCl, CuCl, 37% aqueous HCl;
(b) NaH, Me2CO3; (c) methyl vinyl ketone, NaOMe, MeOH; (d) 2,3-
butanediol, PPTS, toluene; (e) LAH, Et2O; (f) (COCl)2, DMSO,
CH2Cl2; (g) Ohira reagent, K2CO3, MeOH; (h) 10% aqueous HCl,
MeOH; (i) HCO2Et, NaOMe, PhH; (j) NH2OH·HCl, aqueous
EtOH; (k) NaOMe, MeOH, Et2O; (l) PhSeCl, pyridine, CH2Cl2, 30%
aqueous H2O2, CH2Cl2.
a
Reagents: (a) Pd(OAc)2, RuPhos, MeB(OH)2, K3PO4, toluene; (b)
2,3-butanediol, PPTS, toluene; (c) LAH, Et2O; (d) (COCl)2, DMSO,
CH2Cl2; (e) Ohira reagent, K2CO3, MeOH; (f) 10% aqueous HCl,
MeOH; (g) HCO2Et, NaOMe, PhH; (h) NH2OH·HCl, aqueous
EtOH; (i) NaOMe, MeOH; (j) PhSeCl, pyridine, CH2Cl2, 30%
aqueous H2O2, CH2Cl2.
ligand,26,27 RuPhos (2-dicyclohexyl-phosphino-2′,6′-diisopro-
poxybiphenyl), Pd(OAc)2, and K3PO4 for the Suzuki coupling
between 62 and MeB(OH)2. The desired methyltricyclic
compound 67 was obtained from 62 in 80% yield. Ketalization
of 67 with 2,3-butandiol, followed by LAH reduction, gave 68
in 96% yield. The target compound 13 was prepared in 7 steps
from 68 by the same sequence as for 11 and 12 (50% yield).
Tricyclic ethynylcyanodienones 14 and 15, which have
fluorene skeletons, were synthesized in 11 steps from 1-
indanone (69) and 7-chloroindanone (76), respectively
(Scheme 10). Unexpectedly, a previously reported method25
for conversion of 7-amino-1-indanone (74) to 76 using CuCl2
and t-BuNO2 in CH3CN gave 76 in much lower yield (15%)
than the reported yield (64%), with N-(3-oxo-2,3-dihydro-1H-
inden-4-yl)acetamide (75) as the major (side) product.28
However, we could obtain 76 in 86% yield by the traditional
Sandmeyer reaction conditions (diazotization and subsequent
chlorination with CuCl under acidic conditions). Methox-
ycarbonylation and subsequent Robinson annulation of 69 and
76 provided the known compound 7025,29 and the new
compound 77 in 60% and 45% yield, respectively. The ketal of
fluorene 70 with ethylene glycol was easily converted to the
original 70, while the ketal of phenanthrene 56 with ethylene
glycol was stable (see Scheme 7). Enones 70 and 77 were
protected with 2,3-butandiol to give 71 and 78, respectively.
Ethynylenones 73 and 80 were prepared via 72 and 79 from 71
and 78 in four steps (LAH reduction, Swern oxidation,
ethynylation with Ohira reagent, and deprotection under acidic
conditions), respectively. The desired compounds 14 and 15
were obtained in 61% and 45% yield from 73 and 80 in four
steps (formylation, isoxazole formation, introduction of a cyano
group, and insertion of a double bond), respectively.
3. RESULTS AND DISCUSSION
3.1. Chemical Reactivity of Ethynylcyanodienones as
Michael Acceptors. We have evaluated the chemical reactivity
of monocyclic, bicyclic, and tricyclic compounds 4−15 as
Michael acceptors using UV spectroscopy. Michael reaction
between ethynylcyanodienones with DTT give adducts IV
(Scheme 11). Because adducts IV have different UV absorption
spectra (λmax 330−380 nm) from those of the corresponding
ethynylcyanodienones (λmax 230−320 nm), UV spectroscopy
can clarify whether or not the Michael adducts are produced.6
Scheme 11
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a
Table 1. UV Spectra and Biological Potency of Ethynylcyanodienones 3−15
b
without DTT (with
with DTT (10 equiv)
with DTT (1 equiv)
Cl)
d
d
d
c
h
i
compd
λmax (nm)
A
λmax (nm)
A
λmax (nm)
A
K × 103 L/mol
NQO1 CD (nM)
iNOS IC50 (nM)
e
3
4
334
333
250
338
335
337
337
333
382
380
0.406
0.510
0.439
0.481
0.353
0.355
0.472
0.872
0.723
0.262
334
330
0.227
0.614
322
328
0.083
0.635
2.0
21
18
10.6
180
210
5
6
7
8
9
335
332
333
333
331
380
380
0.369
0.135
326
320
324
324
321
0.197
0.036
0.151
0.279
0.831
4.0
1.0
2.7
4.2
20
42
16
29
0.281
56
160
200
NT
230
500
650
940
380
800
1.0
0.376
100
220
150
900
1300
1800
41
g
0.848
NT
g
10
11
12
13
14
15
shoulder
shoulder
NT
g
NT
f
f
f
f
g
NT
NT
NT
NT
NT
g
374
382
383
0.279
0.820
0.803
NT
g
377
378
0.808
0.819
364
369
0.867
1.025
NT
g
NT
130
0.9
TBE-31 (1)
CDDO
2.3
23
sulforaphane
200
400
a
UV spectra of the Michael adducts between ethynylcyanodienones (0.1 mM) and DTT (1 mM and 0.1 mM) in phosphate buffer saline−1%
b
ethanol (pH 7.4) at rt. The full UV spectra of the reaction mixtures are shown in Figure S1 in the Supporting Information. Some
c
ethynylcyanodienones gave Michael adducts with Cl in phosphate buffer saline−1% ethanol (pH 7.4). Equilibrium constant at 0.1 mM of
d
e
f
compound with 0.1 mM of DTT. A: absorbance. See ref 6. Not observed. Compound 12 was not soluble in phosphate buffer saline−1% ethanol
g
(pH 7.4). Equilibrium constant at 0.1 mM of compound with 0.1 mM of DTT could not be obtained because the UV spectrum of the compound is
h
not as simple as that of 3. Hepa1c1c7 cells (10000 per well) were grown in 96-well plates for 24 h and then treated with increasing concentrations
of compounds for 48 h. Cells were lysed, and the protein concentration of the lysates was determined by the bicinchoninic acid (BCA) assay
(Thermo Scientific). The concentration required to double (CD) the specific enzyme activity of NQO1 was used to quantity inducer potency. The
i
value is based on the activity from eight replicate wells at each concentration. The standard deviation in each case was between 5 and 10%. RAW
264.7 cells (20,000 per well) were grown in 96-well plates for 24 h. Cells were then cotreated with LPS (10 ng/mL) and increasing concentrations of
compounds for a further 48 h. The concentration of NO in the cell culture medium was determined using the Griess reagent. The cells were lysed,
and the protein concentration of the lysates was determined by the BCA assay. IC50 values were determined based on suppression of LPS-induced
NO production normalized to the protein concentration. The value is based on determinations from eight replicate wells at each concentration.
Results shown are the average of three independent experiments.
3.1.1. UV Studies on Monocyclic and Bicyclic Compounds
4−9 with DTT. The UV spectra of compounds 5−8 with DTT
are similar to those of 3.6 Compounds 5−8 have local
maximum absorptions at 332−338 nm upon the addition of
DTT (1 and 10 equiv) under dilute (0.1 mM of the
compounds) and neutral aqueous conditions (pH 7.4
phosphate buffered saline−1% ethanol containing 1 mM
KH2PO4, 5.6 mM Na2HPO4, and 154 mM NaCl) (Table 1
and Supporting Information Figure S1). Additionally, these
compounds react with a chloride anion (a much weaker
nucleophile than a sulfhydryl group of DTT), which is
contained in the buffer solution, to give Michael adducts
whose local maximum absorptions are observed at 320−326
nm. We have calculated approximate equilibrium constants (K)
of these Michael reactions in the solutions of 5−8 with DTT
(each initial concentration, 0.1 mM; see the calculation of the
equilibrium constants in the Supporting Information). The
values of K are approximately 1.0−4.2 × 103 (L/mol), implying
that these additions are very strongly favored.
Compound 4 has two local maximum absorptions at 333 and
250 nm (absorbance (A) = 0.510 and 0.439, Table 1 and
Supporting Information Figure S1) with 10 equiv of DTT. We
have never observed such a spectrum with DTT. The
wavelengths of the local maximum absorbance (λmax) at 333
and 250 nm are assigned to those of 81 (cyanodiene) and 82
(cyanoene), respectively (Scheme 12). Similarly, 4 reacts with a
chloride anion, producing a high concentration of the adduct; a
similar high concentration of the adduct was observed with
DTT (1 equiv). Interestingly, these findings indicate that the
two ethynyl groups at C3 increase the reactivity of both
cyanoenone and enone without a cyano group. The
approximate K of the Michael reactions of 4 with DTT (each
initial concentration, 0.1 mM) is 10.6 × 103 (L/mol), and the
value is much higher than those of 3 and 5−8. This means that
the addition of 4 with DTT is much more favored than those of
3 and 5−8.
These K values demonstrate that the order of the reactivity as
Michael acceptors is 4 (ethynyl group) ≫ 8 (dimethylcyclo-
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the ability to activate the Keap1/Nrf2/ARE pathway,
compounds 4−15 were evaluated for the induction of the
classical Nrf2 target, the phase 2 cytoprotective enzyme NQO1
in Hepa1c1c7 murine hepatoma cells.30,31 In this assay, the
concentration required to double (CD value) the specific
enzyme activity of NQO1 is used to quantify inducer potency.
The CD values (nM) of 3−15, TBE-31, and CDDO are shown
in Table 1. The CD values of these compounds are observed in
the 20−2000 nM concentration range. Notably, many of these
compounds are more potent than sulforaphane,32 which is a
widely used activator of the Keap1/Nrf2/ARE pathway and has
demonstrated protective effects in many disease models related
to inflammation and cancer.32,33 Among these compounds, 5 is
the most potent and the potency is similar to that of 3.
Compounds 6 and 14 are next in the rank order of potency.
Interestingly, the bicyclic compound 7 and tricyclic compounds
10, 11, and 14 induce NQO1 more effectively than monocyclic
compounds 3−6 at the higher concentrations (Figure 3)
although these compounds are less potent than the monocyclic
compounds according to their CD values. These compounds
may be more robust inducers than monocyclic compounds.
As previously reported,6 we have found a correlation between
reactivity of monocyclic cyanoenones as Michael acceptors and
biological potency. In this series of monocyclic and bicyclic
compounds 3−9, we found a similar correlation. Compounds 4
and 9, which have the highest reactivity with a sulfhydryl group
and a chloride anion in this series, and compound 8, which has
the third highest reactivity, are less potent than other
compounds which have much lower reactivity with a chloride
anion than 4, 8, and 9. On the basis of these results and our
previous observations,6 we speculate that (i) due to its
exceedingly high chemical reactivity, a large portion of 4, 8,
and 9 could be “quenched” by abundant cellular thiols, such as
the cysteine residue of glutathione, which is present at
millimolar concentrations, (ii) 4, 8, and 9 could be inactivated
by chloride anion in the cell culture medium used for biological
testing, and/or (iii) in addition to the chemical reactivity, other
factors such as cellular uptake and export mechanisms may play
a role in determining the biological potency. Among the
compounds whose reactivity with a chloride anion is relatively
low, interestingly, compound 5, which has the highest reactivity
with a sulfhydryl group, is the most biologically potent.
Scheme 12. Michael Reaction between Compound 4 and 10
equiv of DTT
pentane ring) ≥ 5 (vinyl group) > 7 (cyclopentane ring) > 3
(methyl group) > 6 (ethyl group). We have observed a
tendency that electron-donating groups at the C3 position
decrease the reactivity. Notably, an ethynyl group, a moderate
electron-withdrawing group, enhances the reactivity of enone
without a cyano group. Importantly, the enolizable cyanodie-
none 7 is less reactive than the corresponding nonenolizable
cyanodienone 8.
The naphthalene derivative 9 also reacts with a chloride
anion to give a similar high concentration of the adduct to
those of the adducts with DTT (1 and 10 equiv). The reactivity
of 9 is similar to that of 4.
3.1.2. UV Studies on Tricyclic Compounds 10−15 with
DTT. These tricyclic compounds 10−15 react with DTT to give
the adducts whose local maximum absorptions are observed at
374−383 nm range (Table 1 and Supporting Information
Figure S1). Interestingly, in this series, phenanthrene
derivatives 10−13 are much less reactive than fluorene
derivatives 14 and 15. Phenanthrene derivatives do not react
with a chloride anion, while fluorene derivatives react with a
chloride anion to give similar concentrations of the adducts to
those of the adducts with DTT. Among phenanthrene
derivatives, substituents at C5 decrease the reactivity. Overall,
UV spectra demonstrate that the order of the reactivity as
Michael acceptors is 14 and 15 (fluorene) ≫ 10 (phenan-
threne) > 11 (5-chlorophenanthrene) > 13 (5-methylphenan-
threne).
On the other hand, a series of tricyclic compounds with
phenanthrene and fluorene skeletons, 10−15, show a different
correlation between reactivity and potency. The fluorene
derivatives 14 and 15 are more potent than the phenanthrene
derivatives 10−13, while 14 and 15 show much higher
reactivity with a chloride anion and a sulfhydryl group than
10, 11, and 13. Notably, among these monocyclic, bicyclic, and
3.2. Biological Results. 3.2.1. Compounds 4−15 Induce
NQO1 in Hepa1c1c7 Murine Hepatoma Cells. To determine
Figure 3. Dose response curves of compounds 3−15.
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Article
tricyclic compounds 3−15, 14 shows not only the third lowest
CD value but also induces NQO1 to a greater magnitude than
3 and 5 at higher concentrations. In a series of phenanthrene
derivatives, the potency is well correlated to the reactivity.
3.2.2. Compounds 4−15 Inhibit NO Production Induced
by LPS Stimulation in RAW 264.7 Cells. We evaluated the
inhibitory activities of 3−15 on NO production in RAW 264.7
cells stimulated with LPS.34 The inhibitory activities [IC50
(nM) values] of 3−15, TBE-31, and CDDO are shown in
Table 1. Among these new compounds, 5 has the highest
potency, which is similar to that of 3. Notably, both 3 and 5,
which are monocyclic compounds, are as potent as or even
slightly more potent than a pentacyclic tritepenenoid, CDDO.
We previously demonstrated a linear correlation between
NQO1 inducer potency (CD) and inhibitory activity against
NO production (IC50) of semisynthestic triterpenoids35,36 and
synthetic tricyclic compounds.2 However, we did not observe a
linear correlation in monocyclic cyanoenones which have been
previously reported, although there was a general correlation
between the rank order of potencies in the two assays.6
Remarkably, in this series of compounds 3−13, we observed an
even more striking linear correlation (r2 = 0.96) than the
previous reported correlations (r2 = 0.91)2,35 (Figure 4).
compounds, which have low reactivity with a chloride anion but
high reactivity with a sulfhydryl group, are highly potent as the
inducers and the inhibitors. A monocyclic compound 5 is the
most potent among them and is as potent as the electrophilic
fragment 3. Exceptionally, while 14 has high reactivity with a
chloride anion, 14 has high potency as the inducer of NQO1.
However, potency of 14 as the inhibitor of iNOS is weak.
Notably, we observed a striking linear correlation (r2 = 0.96)
between the potency of the compounds 3−13 as inducers of
NQO1 (CD values) and as inhibitors of iNOS (IC50 values),
but compounds 14 and 15 do not fit this line. As the iNOS
inhibitory activity is only partially dependent on Nrf2,37 this
finding suggests that 14 and 15 may be more specific as Nrf2
activators than as iNOS inhibitors. Although the potency of 14
is lower than that of 3 and 5 according to the CD values, the
magnitude of NQO1 induction by 14 is greater than that by 3
and 5 at concentrations higher than the CD values. Thus, 14
may be a more robust inducer than 3 and 5. Overall,
interestingly and notably, 14 is clearly different from the
other compounds in this series.
We have clarified that the designed compounds containing
the fragment 3 maintain the features of the reactivity and
biological potency of 3. Thus, 3 is a useful electrophilic
fragment for exploring reversible covalent drugs. Indeed,
fragment 3-conatining compound 1 (TBE-31) is highly
bioavailable and extremely potent in protecting against tumor
development in a preclinical model of solar-simulated UV
radiation-mediated cutaneous squamous cell carcinoma.38
Currently, further design and synthesis of reversible covalent
drugs based on the electrophilic fragment 3 are underway.
ASSOCIATED CONTENT
* Supporting Information
■
S
Synthetic procedures and characterization data for new
compounds 1−80, UV spectra of compounds 3−11 and 13−
15 with DTT, and list of elemental analyses for specifying the
purity of 4−15. The Supporting Information is available free of
charge on the ACS Publications website at DOI: 10.1021/
acs.jmedchem.5b00393.
Figure 4. Correlation of potencies of ethynylcyanodienones 3−8, 10−
13 (as shown in blue circle), and 14−15 (shown as orange circles) as
inducers of NQO1 in Hepa1c1c7 murine hepatoma cells, expressed as
CD values, and for suppression of iNOS induction by LPS in RAW
264.7 cells, expressed as IC50 values. The linear correlation coefficient
for the blue circles is r2 = 0.96.
AUTHOR INFORMATION
■
Corresponding Author
*Phone: 631-632-7162. Fax: 631-632-7942. E-mail: tadashi.
honda@stonybrook.edu.
Present Address
Interestingly, the fluorene derivatives 14 and 15 are not
correlated to this line. While 14 has high potency in the NQO1
induction assay, 14 shows only moderate inhibitory activity in
the iNOS assay.
#For S.Z.: School of Pharmaceutical Science, Wenzhou Medical
University, Wenzhou, Zhejiang 325035, China.
Notes
The authors declare no competing financial interest.
4. CONCLUSION
ACKNOWLEDGMENTS
We thank Dr. Vincent Zoete (Swiss Institute of Bioinformatics,
To explore reversible covalent drugs, we have designed,
synthesized, and biologically evaluated new monocyclic,
bicyclic, and tricyclic ethynylcyanodienones 4−15 containing
3 as the electrophilic fragment.
The designed compounds have been synthesized in 7−11
steps and relatively good yields from the starting materials. We
employed the Diels−Alder reaction and Robinson annulation
for the key cyclization steps of these syntheses.
In this series of monocyclic, bicyclic, and tricyclic compounds
excluding a fluorene derivative 14, biological potency of the
compounds, which have high reactivity with a chloride anion,
are weak as inducers of NQO1 and as inhibitors of iNOS. The
■
Switzerland) and Dr. Rene
́
V. Bensasson (Museu
́
m National
d’Histoire Naturelle, France) for helpful discussions and
suggestions. We also thank Dr. Bela Ruzsicska (Stony Brook
University) for expert technical assistance with LC-MS. This
investigation was supported by funds from Stony Brook
Foundation, Reata Pharmaceuticals, the BBSRC (BB/
J007498/1 and BB/L01923X/1), and Cancer Research UK
(C20953/A10270). W.L., S.Z., and C.W.C. are grateful to the
Institute of Chemical Biology & Drug Discovery Postdoctoral
Scholarships.
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ABBREVIATIONS USED
■
ARE, antioxidant response element; CDDO, 2-cyano-3,12-
dioxooleana-1,9(11)-dien-28-oic acid; IFN-γ, interferon-γ;
IKKβ, inhibitor of nuclear factor κB kinase β; iNOS, inducible
nitric oxide synthase; JAK1, Janus kinase 1; Keap1, Kelch-like
ECH-associated protein 1; LPS, lipopolysaccharide; MSK,
mitogen- and stress-activated kinase; Nrf2, nuclear factor-
erythroid 2 p45-related factor 2; p-TsCN, p-toluenesulfonyl
cyanide; RSK, ribosomal s6 kinase
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